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Abstract:

The present invention relates to Rhus verniciflua Stokes extract having
an increased content of an active flavonoid compound through a gas
bubbling treatment, a method for preparing the Rhus verniciflua Stokes
extract, and a method for converting fustin into fisetin through the gas
bubbling treatment of a solution containing fustin. The method for
preparing the Rhus verniciflua Stokes extract of the present invention
can convert the conventional Rhus verniciflua Stokes extract having high
content of fustin and low content of fisetin into a Rhus verniciflua
Stokes extract having high content of fisetin. Therefore, the Rhus
verniciflua Stokes extract of the present invention can be commercialized
into natural anticancer agents, anticancer and cancer prevention food
products, and health functional foods due to the excellent bioactivity
thereof, and promote the development of relevant industries through the
high added-value of Rhus verniciflua Stokes. In addition, the method for
converting fustin into fisetin can be applied to other natural substances
and be used as a method for enhancing the functionality thereof by
increasing the content of a high active flavonoid, thereby becoming an
enabling technology that can lead high-functionalization of natural
substances.

Claims:

1. A method for preparing a Rhus verniciflua stokes extract with an
increased content of an active flavonoid compound, comprising: extracting
Rhus verniciflua stokes extract using an extraction solvent; and
performing a gas bubbling treatment into the Rhus verniciflua stokes
extract.

2. The method of claim 1, wherein the content ratio of fisetin:fustin in
the extract is 1:0 to 2:1.

3. The method of claim 1, wherein the gas bubbling treatment is performed
by using at least one way selected from the group consisting of along
with the extraction of Rhus verniciflua stokes at the same time; after
completing the extraction of Rhus verniciflua stokes; and in the
concentrated solution of the Rhus verniciflua stokes extract.

4. The method of claim 1, wherein the gas includes oxygen.

5. The method of claim 1, wherein the gas is air and the time for the gas
bubbling treatment is 6 to 24 hours.

6. The method of claim 1, wherein the gas is oxygen with high purity, at
least 95% (v/v) and the time for the gas bubbling treatment is 5 to 12
hours.

7. The method of claim 1, wherein the gas bubbling treatment is performed
by at least one method selected from the group consisting of directly
bubbling gas through the extract solution inside an extractor; contacting
gas with the extract solution outside the extractor while mixing; and
contacting gas with the extract solution while passing the extract
solution through a vessel which is filled with gas.

8. The method of claim 1, wherein the active flavonoid is fisetin.

9. Rhus verniciflua stokes extract with an increased content of an active
flavonoid compound prepared by using the method according to claim 1.

11. The Rhus verniciflua stokes extract of claim 10, wherein the content
ratio of fisetin:fustin in the extract is 1:0 to 2:1.

12. An anticancer composition, comprising the Rhus verniciflua stokes
extract according to claim 9 as an effective component.

13. A health functional food for preventing cancer or improving cancer
symptoms, comprising the Rhus verniciflua stokes extract according to
claim 9 as an effective component.

14. A fraction of Rhus verniciflua stokes extract with an increased
content of fisetin, obtained by adding an organic solvent to the Rhus
verniciflua stokes extract according to claim 9, and then fractionating.

15. The fraction of claim 14, wherein the organic solvent is selected
from the group consisting of acetone, ethyl acetate, n-butanol,
chloroform, and combination thereof.

16. The fraction of claim 15, wherein the content of fisetin in the
fraction is within a range of 30 to 45% (w/w).

17. A method of converting fustin into fisetin, comprising performing a
gas bubbling treatment on a solution containing fustin.

18. The method of claim 17, wherein the gas includes oxygen.

19. An anticancer composition, comprising an effective amount of the
fraction according to claim 14 and a pharmaceutically acceptable carrier.

20. A health functional food for preventing cancer or improving cancer
symptoms, comprising a food stuff and an effective amount of the fraction
according to claim 14.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of Korean
Patent Application No. 2010-0077410, filed Aug. 11, 2010, the disclosure
of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] 1. Field of the Invention

[0003] The present invention relates to a method for preparing a Rhus
verniciflua stokes extract having an increased content of an active
flavonoid compound by performing a gas bubbling treatment on the Rhus
verniciflua stokes extract; the Rhus verniciflua stokes extract prepared
by the above method; and a method of converting fustin into fisetin by
performing a gas bubbling treatment on a solution containing fustin.

[0004] 2. Discussion of Related Art

[0005] Rhus verniciflua stokes is fallen leaves broad leaf arborescent
belonging to the Anacardiaceae family and it is known that Rhus
verniciflua stokes is originally from the Himalayas and highlands of
Central Asia. It is now distributed widely throughout the world, from the
subtropics to the temperate regions, with the tropics as the center. Rhus
verniciflua stokes is fallen leaves or evergreen trees and most of them
are shrub trees or tall trees, but some of them are climbing trees
(Barkley Fred Alexander., Ann. of the Missouri Bot. Garden., 24(3), pp
265-500, 1937).

[0006] A sap of Rhus verniciflua stokes is known as an oriental lacquer.
In terms of oriental medicine, it is widely known that a dried oriental
lacquer removes extravasated blood; improves blood circulation; and is
effective in reducing or alleviating hookworm, stomachache, excessive
acid in the stomach, thick sputum, tuberculosis, period pains,
constipation, diabetes, malaria, anti-inflammatory, arthritis, and is
useful for use as a preservative, strengthening the stomach, easing
menstruation pain, and the like, and it is recently known that it is
effective in preventing cancer (Namba, T., Colored Illustrations of Wakan
Yaku. p 215, Hoikusha Publishing Co. Ltd., Osaka, 1980). In addition, in
terms of common medicine, it is known that it is useful as a digestive
medicine; it controls extravasated blood in the liver; it controls
cardiac disease as a drug for cleaning blood in the heart; removes
tuberculosis germs in the lung; and also it is an excellent drug for
neuralgia, arthritis, skin diseases, and the like because Rhus
verniciflua stokes and a wild ginseng are comparable to each other in
effect.

[0007] It is already known from much research that Rhus verniciflua stokes
include a lot of anti-oxidative substances. Specifically, it is known
that the Rhus verniciflua stokes extract that is extracted by using
ethanol has strong anti-oxidative activity and also the fraction that is
isolated and purified by using a silica column has an ability for
inhibiting the growth of tumor cells in the human blood. In addition, it
is also known that a substance having an antimicrobial effect is isolated
from the Rhus verniciflua stokes extract that is extracted by using
ethanol and Rhus verniciflua stokes skin extract that is extracted by
using methanol has various bioactivity effects related to the Rhus
verniciflua stokes extract, such as an effect on suppressing obesity.

[0009] The above components are mostly flavonoid-based materials and
flavonoids, such as fisetin and fustin, which play a role in protecting
blood vessels or capillaries. And also, the fisetin and fustin are very
excellent bioactive substances having antioxidative activity,
anti-inflammatory properties, and anticancer properties. However, the
fustin that is plentifully included in the Rhus verniciflua stokes
extract has a disadvantage wherein its activity is greatly decreased as
compared to the activity of fisetin in terms of bioactivity. On the other
hand, the fisetin has activitystrong anticancer activity so that it is
now being used as an anticancer drug, but there is a disadvantage that a
major component of most conventional Rhus verniciflua stokes extracts is
the fustin and the fisetin is included in small quantity as compared to
the fustin.

[0010] Accordingly, when the Rhus verniciflua stokes extract containing a
lot of fisetin with excellent bioactivity may be prepared, the Rhus
verniciflua stokes extract may have relatively excellent anticancer
activity and bioactivity so that it can be expected to be very useful in
the food and medicine industries.

SUMMARY OF THE INVENTION

[0011] Accordingly, the present inventors preformed research into a method
for preparing a Rhus verniciflua stokes extract having very excellent
bioactivity by controlling the content of a flavonoid compound in the
Rhus verniciflua stokes extract.

[0012] Therefore, the present inventors completed the present invention by
developing a method for preparing new Rhus verniciflua stokes extract
including a large quantity of fisetin having various bioactivities, as
well as anti-oxidative activities and anticancer activities.

[0013] Accordingly, it is an object of the present invention to provide
Rhus verniciflua stokes extract with an increased content of an active
flavonoid compound by converting fustin, a flavonoid compound, into
fisetin, an active flavonoid compound, in which the active flavonoid
compound is plentifully included in the Rhus verniciflua stokes extract.

[0014] In order to achieve the above objects, there is provided a method
for preparing a Rhus verniciflua stokes extract with an increased content
of an active flavonoid compound, including: extracting Rhus verniciflua
stokes extract using an extraction solvent; and performing a gas bubbling
treatment into the Rhus verniciflua stokes extract.

[0015] In an embodiment of the present invention, the gas may include
oxygen.

[0016] In an embodiment of the present invention, the gas bubbling
treatment may be performed by using at least one way of implementation
thereof along with the extraction of Rhus verniciflua stokes at the same
time, or after completing the extraction of Rhus verniciflua stokes.
Preferably, the gas bubbling treatment may be performed by at least one
method of directly bubbling gas through the extract solution inside an
extractor, or contacting gas with the extract solution outside the
extractor.

[0017] In an embodiment of the present invention, the active flavonoid is
fisetin.

[0019] In an embodiment of the present invention, the gas may be air and
the time for the gas bubbling treatment may be 6 to 24 hours. Preferably,
the time for the gas bubbling treatment may be 8 to 12 hours.

[0020] In an embodiment of the present invention, the gas may be oxygen
with high purity, preferably at least 95% (v/v), and more preferably pure
oxygen, and the time for the gas bubbling treatment may be 5 to 12 hours.
Preferably, the time for the gas bubbling treatment may be 7 to 10 hours.

[0021] In addition, the present invention provides a Rhus verniciflua
stokes extract including fisetin prepared by performing a gas bubbling
treatment, wherein the content ratio of fisetin:fustin in the extract is
1:0 to 2:1. This means that the content ratio value of fisetin/fustin is
2 or more.

[0022] In addition, the present invention provides a method of converting
fustin into fisetin, including performing a gas bubbling treatment into a
solution containing fustin.

[0023] In an embodiment of the present invention, the gas may be air and
the time for the gas bubbling treatment may be 6 to 24 hours. Preferably,
the time for the gas bubbling treatment may be 8 to 12 hours.

[0024] In an embodiment of the present invention, the gas may be oxygen
with high purity, preferably at least 95% (v/v), and more preferably pure
oxygen, and the time for the gas bubbling treatment may be 5 to 12 hours.
Preferably, the time for the gas bubbling treatment may be 7 to 10 hours.

[0025] In addition, the present invention provides a Rhus verniciflua
stokes extract including fisetin, wherein the content ratio of
fisetin:fustin is 1:0 to 2:1.

[0026] In addition, the present invention provides a pharmaceutical
composition including the Rhus verniciflua stokes extract as an effective
component for preventing or treating cancer.

[0027] In addition, the present invention provides a health functional
food including the Rhus verniciflua stokes extract as an effective
component for preventing cancer or improving cancer symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and other objects, features and advantages of the present
invention will become more apparent to those of ordinary skill in the art
by describing in detail exemplary embodiments thereof with reference to
the attached drawings, in which:

[0029] FIG. 1 is a graph showing an effect on converting fustin into
fisetin according to time when air bubbling treatment is applied to Rhus
verniciflua stokes extract; and

[0030]FIG. 2 is a graph showing an effect on converting fustin into
fisetin according to time when high purity oxygen bubbling treatment is
applied to Rhus verniciflua stokes extract.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0031] The present invention provides a method for preparing a Rhus
verniciflua stokes extract having an increased content of an active
flavonoid compound, including extracting Rhus verniciflua stokes extract
using an extraction solvent; and performing a gas bubbling treatment into
the Rhus verniciflua stokes extract.

[0033] In Rhus verniciflua stokes, the content of flavonoid component that
is known as a medical component is increased with tree age. Generally,
only when the tree age is over 10 years, the content of flavonoid may be
included in about 15 to 20% (w/w) among the extract based on the extract.
13 to 17% (w/w) among them is fustin (2,3-Dihydrofisetin) represented by
Chemical Formula 1, 1 to 3% (w/w) is fisetin represented by Chemical
Formula 2, and then trace amounts of sulfuretin, butein, and the like are
included.

##STR00001##

##STR00002##

[0034] It is known that fustin that is plentifully included in Rhus
verniciflua stokes extract has antioxidative activity, anticancer
activity, etc. like fisetin, but useful bioactivity of fustin is greatly
decreased as compared to that of fisetin. The comparison experiment for
antioxidative activities between fustin and fisetin that was confirmed by
the present inventors supports the above fact (see Table 1). That is,
according to an embodiment of the present invention, it has been seen
that the antioxidative activity of fisetin was excellent by at least 5
times as compared with that of fustin as a result of the comparison
experiment for anti-oxidative activities between fustin and fisetin.

[0035] Accordingly, since fustin has relatively low bioactivity effect as
compared with that of fisetin that is an active flavonoid having
excellent anticancer and anti-oxidative activities, fustin refers to a
"non-active flavonoid" for the present invention.

[0036] Therefore, the present invention provides a method for preparing a
Rhus verniciflua stokes extract by converting fustin that is plentifully
included in Rhus verniciflua stokes extract into fisetin that is an
active flavonoid with excellent bioactivity, and the Rhus verniciflua
stokes extract prepared according to the present invention includes a
large quantity of an active flavonoid compound.

[0037] The method for preparing the Rhus verniciflua stokes extract
according to the present invention includes performing a gas bubbling
treatment on Rhus verniciflua stokes extract.

[0038] The Rhus verniciflua stokes refers to the trees belonging to the
Anacardiaceae family, and a type of useable Rhus verniciflua stokes may
include any one or more selected from the group consisting of Rhus
trichocarpa MIQ, Rhus verniciflua, Rhus cotinus, Rhus ambigua H.Lev.,
Succedanea Sumac, and Rhus succedanea L., but the present invention is
not limited thereto. Preferably, Rhus verniciflua may be used.

[0039] A proper solvent for extracting Rhus verniciflua stokes extract
according to the present invention may include water and an organic
solvent, and preferably may include alone or a combination of various
solvents, such as water, alcoholic solvent, methanol, ethanol, propanol,
isopropanol, butanol, acetone, ether, benzene, chloroform, ethylacetate,
methylenechloride, n-hexane, hydrochloric acid, acetic acid, formic acid,
citric acid, cyclohexane, petroleum ether, and the like. More preferably,
water, an alcoholic solvent, and methanol may be used, and most
preferably, water may be used.

[0040] The Rhus verniciflua stokes extract according to the present
invention may be prepared by a general method for preparing an extract,
and specifically, may be prepared by a hot-water extraction method, a
macerating extraction method, a digesting extraction method, and the
like, and may be prepared by using a general extracting machine,
sonicator, or fractionators. In addition, the extract is extracted by
using a solvent and then filtering, concentrating, or drying may be
selectively performed to remove the solvent from the extract or all of
filtering, concentrating, and drying may be performed to remove the
solvent from the extract. Specifically, the filtering may include
decompression filtering or pressurization filtering using a filter for
filtering, and the concentrating may include decompression concentrating
in a vacuum. In addition, water of the resulting extract may be
completely removed through the concentrating and drying, and the Rhus
verniciflua stokes extract without water may be used in a type of powder
or the powder may be solved in distilled water or general solvent to use.
Accordingly, the Rhus verniciflua stokes extract including a high content
of an active flavonoid compound that is obtained by extracting and
converting the Rhus verniciflua stokes extract according to the present
invention may be commercialized as an extract powder or extract solution
through filtering, concentrating, or drying process.

[0041] In addition, useable gas in the bubbling treatment may include gas
containing oxygen, if it does not disrupt the reaction, preferably, high
purity oxygen, and more preferably, pure oxygen, but the present
invention is not limited thereto.

[0042] A method for preparing the Rhus verniciflua stokes extract with an
increased content of an active flavonoid compound according to the
present invention are described in more detail, as follows:

[0043] Water, alcoholic solvent, an organic solvent, or diluents solution
thereof is added as a solvent for extracting Rhus verniciflua stokes, and
then is heated to extract. In the extracting process, gas, preferably the
gas containing oxygen is bubbled through the extraction solution or
concentration solution. The above process is performed to convert fustin,
a non-active flavonoid compound into fisetin, active flavonoid, in which
the fustin is plentifully included in the extract extracted from Rhus
verniciflua stokes. The gas bubbling process may be performed with
extracting at the same time, after extracting for a certain time, or
after making the concentration solution through the concentrating of the
extraction solution that is completely extracted.

[0044] The bubbling gas may include a gas containing oxygen, preferably,
air or high purity air, and preferably, pure oxygen. When the purity
degree of oxygen is high, it has an advantage that even though the
processing time is short; the same effect can be obtained. The purity of
oxygen included in the bubbling gas is 20% (v/v) in the case of air.
According to a method of supplying high purity oxygen with at least 95%
(v/v) purity, the higher the purity of oxygen, the shorter the time
required for converting fustin into fisetin; and the lower the purity of
oxygen, the longer the time required thereby requiring a considerable
time for completely converting.

[0045] When the bubbling treatment is performed for a short time, less
than 1 hour, fustin is only slightly or very slightly converted into
fisetin so that there is a Lag-time, in which the components are not
changed for a period of time, such that a proper gas bubbling treatment
time is required. On the other hand, excessive oxygen bubbling causes
decomposition after converting fustin into fisetin so that it is
important that a proper time should be selected.

[0046] A case of applying the similar method as the method of the present
invention to remove an allergy inducing material is disclosed in Korean
Patent No. 10-0918326, but there is no technology for converting fustin
into fisetin with high activity. The removal of an allergy inducing
material is performed along with a supply of oxygen at the same time and
completed within a short time, while the conversion of fustin into
fisetin is gradually preformed at the end of completing the removal of
the allergy inducing material. Accordingly, the conversion of fustin into
fisetin may be a new technological idea that is not disclosed or has not
been invented in the conventional art for a processing condition thereof.

[0047] When the gas for bubbling is air, it is preferable that the air
bubbling treatment is continuously maintained for 6 to 24 hours, and more
preferably for 8 to 12 hours in order to prepare Rhus verniciflua stokes
extract with an increased content of an active flavonoid compound
according to the present invention.

[0048] In addition, when the gas for bubbling is high purity oxygen, it is
preferable that the bubbling treatment is continuously maintained for 5
to 12 hours, and more preferably for 7 to 10 hours in order to prepare
Rhus verniciflua stokes extract with an increased content of an active
flavonoid compound according to the present invention.

[0049] The amount and time for bubbling oxygen may be generally based on
volumetric flow rate (1/min) of gas, and preferably, unit volumetric flow
rate (VVM, 1/min/volume) which is the volumetric flow rate divided by a
volume of solution. The time for supplying of air may be 1 to 40 hours
depending on a gas supplying flow. However, when the time passes 12
hours, most of fustin is converted into fisetin, such that further
supplying of air is not substantially need. Of course, when the supply
rate of gas is very slow, the time for completely converting into fisetin
may require at least 20 hours. Accordingly, the conversion rate of fustin
into fisetin may be possibly controlled by changing a flow rate of gas or
the time for supplying gas.

[0050] Therefore, the present invention may provide Rhus verniciflua
stokes extract with an increased content of an active flavonoid compound
due to the gas bubbling treatment, and also provide Rhus verniciflua
stokes extract, in which 45 to 100% of the conventional content of fustin
presented in Rhus verniciflua stokes extract may be converted into
fisetin. That is, all of fustin that is an essential component of the
conventional Rhus verniciflua stokes extract can be substantially
converted into fisetin through a method of extracting Rhus verniciflua
stokes according to the present invention so that there may be
substantially no fustin in the Rhus verniciflua stokes extract according
to the present invention.

[0051] Therefore, the present invention may provide Rhus verniciflua
stokes extract having the content ratio of 1:0 to 2:1 of fisetin:fustin.

[0052] The bubbling condition of gas through Rhus verniciflua stokes
extract is varied according to the pressure or purity of the used gas.
That is, in relation to the pressure, the bubbling may be continuously
performed on the bottom of an extractor through a blower, etc. under
atmospheric pressure, or may be performed by supplying high-pressure gas.
It may not be affected by the internal pressure of the extractor. That
is, even when the internal pressure of the extractor or concentrator is
maintained within a range of 0 to 10 atmospheric pressures, the effect
may not be changed. The greater the pressure, the greater the solubility
of oxygen is increased so that the processing time may be reduced, but
economic feasibility and stability may be significantly reduced due to a
sharp increase of installation cost in order to maintain high pressure.

[0053] For a method for preparing a Rhus verniciflua stokes extract
according to the present invention, the gas bubbling may be generally
disclosed in the case of supplying at the bottom of the extractor, but
may be performed by using a way of supplying gas through an appropriate
pipe after installing the appropriate pipe inside the extractor. Also,
the gas bubbling may be performed by a way of separately supplying a gas
to allow for contact between the extract solution and the bubbling gas
outside the extractor. Here, it may be a general system of contacting
liquid and gas, and the system may include a wider area that can allow
for contact between the gas and extract solution as a separate space or
in the upper of the extractor. Accordingly, the gas bubbling treatment
according to the present invention may include a method of bubbling gas
through the extract solution of Rhus verniciflua stokes inside the
extractor that is used for extracting, a method of contacting the
bubbling gas and the extract solution outside the extractor, a method of
flowing the gas and extract solution in the same directions or reverse
directions at the same time, or a method of using an apparatus, such as
an inline mixer, as a machine for mixing the gas and extract solution.

[0054] The Rhus verniciflua stokes extract prepared by the method of the
present invention may be reconstituted as a fractional material with a
high content of fisetin through a further fractionation process. That is,
when the solution fractionation may be performed by using acetone, ethyl
acetate, n-butanol, chloroform, and the like, as organic solvent, the
fractional material that is improved by 30 to 45% (w/w) of fisetin
content may be prepared from the extract with 5 to 25% (w/w) of fisetin
content. In addition, the purified material with at least 90% (w/w)
purity of fisetin may be obtained by performing re-crystallization of the
same. However, when the method for preparing a Rhus verniciflua stokes
extract according to the present invention is not applied, the fisetin
with high purity may not be easily obtained by fractionation and/or
re-crystallization due to an interference of fustin even though the
organic solvent is used. Accordingly, in case of performing the
fractionation and/or re-crystallization according to the inventive
conversion method, there is provided an effective way of obtaining
plentiful amounts of high purity fisetin.

[0055] Generally, a method for preparing a Rhus verniciflua stokes extract
may include adding water, alcoholic solvent, or diluted alcoholic solvent
to Rhus verniciflua stokes, extracting the same, and then concentrating
and/or drying the same. In this case, the composition of flavonoid in the
Rhus verniciflua stokes extract may be composed of 10 to 30% (w/w) of
fustin, 1 to 4% (w/w) of fisetin, and 0.1 to 2%(w/w) of other flavonoid
content. In the case of flavonoid, the content is increased with tree
age. The rate of fustin and fisetin in Rhus verniciflua stokes may be
usually maintained within a constant range, and the whole content is
increased with tree age. In this case, the content ratio of
fisetin:fustin may be from the minimum 1:2.5 to the maximum 1:30, and
generally 1:5 to 1:15.

[0056] Like this, the content ratio of fisetin:fustin may be generally in
the range of 1:2.5 to 30. However, in the case of Rhus verniciflua stokes
extract according to the inventive method for preparing a Rhus
verniciflua stokes extract, the content ratio of fisetin:fustin may be
adjusted low by less than 1:0.2. Generally, the difference of activities
may be great since the content ratio of fisetin:fustin of 1:1, and it may
preferably be 5:1, which means that the content of fisetin is 5 times the
content of fustin and also the content of fisetin is significantly
increased as compared with the conventional Rhus verniciflua stokes
extract. In this case, the content of polyphenol that is known to have
bioactivity, such as anti-oxidative activity, immunity-boosting
properties, and the like is also very highly maintained at 40 to 70%
(w/w).

[0057] When fustin is substantially and completely converted into fisetin
by supplying high purity oxygen, the content ratio of fisetin:fustin may
be maintained at a level of 1:0.001. It means that almost all of fustin
is converted into fisetin and the Rhus verniciflua stokes extract with
high content of fisetin obtained from the above process may become a Rhus
verniciflua stokes extract having high bioactivity.

[0058] Accordingly, as compared with the conventional Rhus verniciflua
stokes extract, the Rhus verniciflua stokes extract having an increased
content of active flavonoid compounds according to the present invention
may have excellent bioactivities, such as anticancer activity,
anti-dementia activity, an improvement of memory, an improvement of
arthritis, anti-inflammatory activity, anti-oxidative activity, an
improvement of blood circulation, and the like.

[0059] According to an embodiment of the present invention, as compared
with the conventional Rhus verniciflua stokes extract, it has been seen
that the Rhus verniciflua stokes extract prepared through the inventive
preparation method has very excellent anticancer and anti-oxidative
activities (see Examples 3 and 4). Especially, with reference to the
anticancer activity of the Rhus verniciflua stokes extract according to
the present invention, according to an embodiment of the present
invention, it has been seen as a result of comparing the effects on
inhibiting growth of cancer cells in mice injected with of the
conventional Rhus verniciflua stokes extract and the Rhus verniciflua
stokes extract of the present invention that the weight and volume of
cancer in the mice injected with the Rhus verniciflua stokes extract
having a large quantity of fisetin according to the present invention are
significantly decreased as compared with the mice injected with the
conventional Rhus verniciflua stokes extract (see Table 3). The above
result supports the reported excellent anticancer activity of the Rhus
verniciflua stokes extract according to the present invention having a
large quantity of fisetin that is known to have excellent anticancer
activity.

[0060] Therefore, the present invention may provide an anticancer
composition including the Rhus verniciflua stokes extract with the
content ratio of fisetin:fustin of 1:0 to 2:1 as an effective component,
and the composition according to the present invention may be used for
treating and preventing cancer as a pharmaceutical composition. In
addition, the anticancer composition according to the present invention
may include 0.1 wt % to 100 wt % of the Rhus verniciflua stokes extract
based on total weight of the composition.

[0061] A type of cancer that can be treated with the composition according
to the present invention may be liver cancer, stomach cancer, colorectal
cancer, lung cancer, breast cancer, rectal cancer, leukemia, pancreatic
cancer, and the like, but the present invention is not limited thereto.

[0062] The anticancer composition according to the present invention may
include alone a pharmaceutically effective amount of the Rhus verniciflua
stokes extract, or at least one of a pharmaceutically acceptable carrier,
excipient, or diluents. The term "a pharmaceutically effective amount"
refers to a sufficient amount for preventing, improving and/or treating
cancer symptoms.

[0063] A pharmaceutically effective amount of the Rhus verniciflua stokes
extract according to the present invention may be 0.5 to 100
mg/day/weight kg, and preferably 0.5 to 5 mg/day/weight kg. However, the
pharmaceutically effective amount may be appropriately changed according
to a degree of cancer symptoms, an age of patient, a weight of patient, a
health condition of patient, a sex of patient, an administering route, a
period of treatment, and the like.

[0064] The expression "pharmaceutically acceptable" refers that it may be
physiologically acceptable, and a side effect or the similar effect
thereof, such as a gastroenteric trouble, and dizziness, may not
generally be caused when administering to humans. Examples of the
carrier, excipient, and diluents may include lactose, dextrose, sucrose,
sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber,
alginate, gelatin, calcium phosphate, calcium silicate, cellulose,
methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate,
propyl hydroxybenzoate, talc, magnesium stearate, and minerals. In
addition, they may further include a filler, anticoagulants, lubricant,
wetting agent, flavoring, emulsifying agent, preservatives, and the like.

[0065] In addition, the composition of the present invention may be
formulated by using a method that is known in the art in order to provide
a rapid, continuous, or delayed release type of an active component after
administering to a mammal. The dosage form may be a type of powder,
granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule,
a sterile injection solution, or a sterile powder.

[0066] The anticancer composition according to the present invention may
be administered through various routes, such as oral, a percutaneous, a
subcutaneous, an intravenous, or an intramuscular injection. The dosage
of active component may be appropriately selected according to various
factors, such as an administering route, an age, a sex, and a weight of
patient, severity of patient, and the like. In addition, the anticancer
composition of the present invention may be administered along with the
compounds that are known to have an effect on preventing, improving, or
treating cancer symptoms. Accordingly, the present invention may provide
a medicine for preventing and/or treating cancer symptoms, in which the
medicine includes the Rhus verniciflua stokes extract according to the
present invention.

[0067] Moreover, the anticancer composition according to the present
invention may provide an effect on relieving cancer symptoms through
functions of excellent anticancer and antioxidative activities, and also
since it may be added to food in order to prevent or improve cancer
symptoms, the composition of the present invention may be used for food
in order to prevent and improve cancer symptoms. Accordingly, the
composition of the present invention may be useful in foods having
effects on preventing and improving cancer symptoms, such as, main raw
materials or added raw materials of foods, food additives, functional
foods or beverages.

[0068] According to the present invention, the term "food" refers to a
natural material or processed material that may include one or more
nutrients, and preferably to be eaten after some processing. As a general
meaning, it may refer that all of foods, food additives, functional
foods, and beverages are included.

[0070] In addition, the term "functional food" refers to a food group of
giving a high added value to food function and expresses a function of a
relevant food for a particular purpose using physical, biochemistry,
and/or biotechnology methods, and the like and also a processed food by
designing for sufficiently expressing a body controlling function related
to a body defense mechanism, prevention and recovery of disease, and the
like, which are functions of food. Specifically, it may be a health
functional food. The functional food may include a cytologically
acceptable food supplement and may further include an appropriate
carrier, excipient, and diluents that are generally used for preparing a
functional food.

[0071] In addition, the term "beverage" refers to the generic term for
drinking for quenching thirst or enjoying taste, and may include a
functional beverage. The beverage may include a composition with the
disclosed rate for preventing and improving anticancer symptoms as an
essential component, and other components, without any special
limitation, and also may include various flavouring agents, natural
carbohydrates, and the like, as a further component, like the general
beverage.

[0072] In addition to the above disclosed components, the food including
the composition according to the present invention may include various
nutritional supplements, vitamins, minerals (electrolyte), flavouring
agents, such as synthetic flavouring agents, natural flavouring agents,
and the like, colorings, fillers (cheese, chocolates, etc.), pectic acid
and salts thereof, alginic acid and salt thereof, organic acid,
protective colloid thickener, pH control agent, stabilizer,
preservatives, glycerin, alcohol, carbonation agent that is used for a
carbonated drink, and the like, and the above components may be used
alone or in combination.

[0073] For the food including the anticancer composition of the present
invention, the amount of the composition of the present invention may be
included in 0.001 wt % to 100 wt %, and preferably, 0.1 wt % to 40 wt %.
For the beverage, it may be included in the range of 0.001 g to 5 g, and
preferably 0.01 g to 2 g based on 100 ml, but it may be below the above
ranges when it is taken for a long time for improving health and hygiene,
and for managing health. Since, an effective component has no problem in
terms of stability, it may be used in the amount of the above ranges, and
the present invention is not limited thereto.

[0074] Therefore, the present invention may provide a health functional
food including the Rhus verniciflua stokes extract as an effective
component according to the present invention for preventing cancer or
improving cancer symptoms, and a type of the food may be powder, granule,
tablet, capsule, or drink, but the present invention is not limited
thereto.

[0075] On the other hand, a method for preparing a Rhus verniciflua stokes
extract having an increased content of an active flavonoid compound
according to the present invention may convert a non-active flavonoid
compound having a relatively low bioactivity into an active flavonoid
compound having excellent bioactivity as mentioned above. At this point,
the non-active flavonoid compound may be fustin and the active flavonoid
compound may be fisetin. Accordingly, the present invention may provide
the method of converting fustin into fisetin.

[0076] The method of converting fustin into fisetin may include performing
a gas bubbling treatment on a solution containing fustin and the gas
bubbling treatment may be performed by bubbling gas through an extract
solution inside the extractor or contacting bubbling gas with the extract
solution outside the extractor. The solution containing fustin may be an
extract extracted from a natural material; and it may be a solution or
extract containing fustin and fisetin at the same time; or it may be a
solution or extract containing fustin only.

[0077] In addition, when the gas for bubbling is air, preferably, the
bubbling treatment may be continuously maintained for 6 to 24 hours and
more preferably, for 8 to 12 hours in order to prepare the solution
containing high content of fisetin through the method of converting
fustin into fisetin according to the present invention.

[0078] On the other hand, when the gas for bubbling is high purity oxygen,
preferably, the bubbling treatment may be continuously maintained for 5
to 12 hours and more preferably, for 7 to 10 hours in order to prepare
the solution containing high content of fisetin through the method of
converting fustin into fisetin according to the present invention.

[0079] Accordingly, the present invention may convert the extract or
solution having a large quantity of fustin into the extract or solution
having a large quantity of fisetin that is an active flavonoid compound
with excellent bioactivity.

[0080] Hereinafter, the present invention will be described in more detail
with reference to the following Examples.

[0081] However, the following Examples are only for illustrating the
present invention, and the content of the present invention is not
limited to the following Examples.

Example 1

Preparation of Rhus Verniciflua Stokes Extract Through Gas Bubbling

[0082] The contents of fustin and fisetin that are included in Rhus
verniciflua stokes extract were analyzed using HPLC. The standard
products were purchased from Sigma Aldrich. As conditions of analyzing by
HPLC, UV of 254 nm was used as a detector and 5% mixing solution of
acetic acid:methanol:acetonitrile=70:20:10 was used for a moving phase.
Total of 10 ul was injected at the flow rate of 1 ml/min. C18 (250
mm*4.6 mm, YMC Pack) was used as a column. An analysis of polyphenol was
performed by using tannic acid as a standard material with
Folin-ciocalteu's phenol reagent. An absorbance was measured at 725 nm
after developing color, and calculated using an external calibration
method with a standard material.

[0084] 100 l of water was added to 10 kg of Rhus verniciflua stokes and
then extracted at 95° C. for 6 hours. When a large quantity of
flavonoid component was included in the extract after 4 hours during
extraction, air (rate of oxygen: 20% (v/v)) was bubbled at the bottom of
the extractor at the rate of 1.5 l/min. The time for bubbling was totally
12 hours and the components were analyzed by collecting a sample every
other hour.

[0085] As a result, the content of fustin was significantly decreased as
time passes and the content of fisetin was increased (FIG. 1). After 11.5
hours, substantially 100% of fustin was converted into fisetin. The Rhus
verniciflua stokes extract was filtered, vacuum-concentrated, and then
powderized to obtain 0.56 kg of brown dry powder; and the content of
fustin in the extract powder that is a final product was 0.01% (w/w); the
content of fisetin was 16.7% (w/w); and the content of polyphenol was
56.4% (w/w). That is, the present inventors may obtain the extract, in
which all of fustin having low activity was converted into fisetin, an
active flavonoid, through a method for preparing a Rhus verniciflua
stokes extract according to the present invention.

[0087] As disclosed in the above Example, <1-1>, 100 l of water was
added to 10 kg of Rhus verniciflua stokes and then Rhus verniciflua
stokes was extracted at 95° C. When a large quantity of flavonoid
component was included in the extract through 4 hours of the extraction
time, high purity oxygen (purity: 95% (v/v)) was bubbled at the bottom of
the extractor at the rate of 1.2 l/min using an oxygen cylinder. The time
for bubbling was totally 12 hours and the components were analyzed by
collecting a sample every other hour.

[0088] As a result, the content of fustin was significantly decreased as
time passes and the content of fisetin was increased according to the
decrease of fustin content (FIG. 2). At 8.8 hours of treatment time,
substantially 100% of fustin was converted into fisetin. The rate of
converting was rapid and the conversion was completely processed as
compared with the use of air. In addition, when the bubbling time is more
than necessary, the content of fisetin prepared by converting fustin was
slightly reduced so that it can be known that the proper treating time
may be important. The extract was filtered, vacuum-concentrated, and then
powderized to obtain 0.55 kg of brown dry powder. The content of fustin
in the extract powder that is a final product was 0.00% (w/w); the
content of fisetin was 12.9% (w/w); and the content of polyphenol was
58.2%.

Comparative Example 1

Preparation of General Rhus Verniciflua Stokes Extract

[0089] 100 l of water was added to 10 kg of Rhus verniciflua stokes and
then extracted while it was heated at 95° C. for 6 hours. The
resulting extract was filtered, vacuum-concentrated, and then powderized
to obtain 0.57 kg of brown dry powder. The content of fustin in the
extract powder that is a final product was 17.3% (w/w); and the content
of fisetin was 1.63% (w/w). The content ratio of fisetin:fustin that were
included in Rhus verniciflua stokes extract through a method for
preparing the conventional Rhus verniciflua stokes extract was 1:10.6 and
the content of polyphenol was 53.1%. That is, in the case of Rhus
verniciflua stokes extract through a common method of extracting without
a bubbling treatment, the content of fustin was at least 10 times higher
than that of the content of fisetin.

Example 2

Preparation of Rhus Verniciflua Stokes Extract Using Various Solvents

[0090] The Rhus verniciflua stokes extracts according to the present
invention were prepared by using various extraction solvents. To achieve
this, purified water, 50% alcoholic solvent, 80% alcoholic solvent, 80%
methanol, and 100% alcoholic solvent were used as an extraction solvent.
The conversion rate of fustin into fisetin according to hours during
extraction process using each of extraction solvents was investigated and
then the times for converting 90% were compared each other. The Rhus
verniciflua stokes extracts were extracted at 95° C. by adding the
extraction solvents that were 10 times to 10 kg of Rhus verniciflua
stokes as disclosed in the above Example. And then the conversion rate
into fisetin was investigated by sampling the extract every other hour
while bubbling high purity oxygen at the bottom of an extractor in 0.3
VVM (volume/volume/min). The results are shown in the following Table 2.

[0091] As shown in the above Table 2, it has been seen that for the
extract prepared by using the method for preparing the Rhus verniciflua
stokes extract according to the present invention, the fustin in the
extract can be converted into fisetin by performing a gas bubbling
treatment regardless of a type of used solvents. However, when the
content of water in the solvent was decreased, the processing time was
increased. Accordingly, from the above result, the present inventors can
confirm that the processing time should be controlled in order to
increase the conversion rate into fisetin according to a type of solvent
in the extraction conditions.

Example 3

Analysis of Anticancer Activity of Rhus Verniciflua Stokes Extract
According to the Present Invention

[0092] In order to analyze anticancer activity of the Rhus verniciflua
stokes extract according to the present invention, firstly the anticancer
activities of Rhus verniciflua stokes extract prepared by the general
method of extracting Rhus verniciflua stokes in the above Comparative
Example 1, the Rhus verniciflua stokes extract according to the present
invention prepared from Example <1-1>, and Sample, "A" of Example 6
were mutually compared.

[0093] The analysis of anticancer activity were performed by using a
comparison of effect on inhibiting a growth of cancer cell after orally
administrating the Rhus verniciflua stokes extract according to the
present invention using a mouse. 5×105 cells of human
non-small cell lung cancer cell line, A549, were subcutaneously injected
to a nude mouse (Immunodeficient mice (male), CanN. Cg-Foxn1nu/CrljBgi).
7 nude mice were assigned for one group after dividing a control group,
the group of Comparative Example 1 and the group of Example <1-1>.
The extract was not administrated to the control group, the conventional
Rhus verniciflua stokes extract extracted from Comparative Example 1 was
administrated to the group of Comparative Example 1, and the Rhus
verniciflua stokes extract according to the present invention extracted
in Example <1-1> was administrated to the group of Example
<1-1>. 300 mg/kg volume was orally administrated for 24 days. A
tumor volume was calculated by using the following formula (A=Long
length, B=Short length) after measuring the long length and short length
of the tumor using Caliper two times per one week during an
administration period; then at 27 days, the tumor was removed and then
its weight was measured.

V ( Tumor Size , mean tumor volume , mm 3
) = AB 2 2 ##EQU00001##

[0094] As a result, as shown in Table 3, it has been seen that the tumor
weight and volume in the mouse of Example <1-1> group were
significantly small as compared with the animal model group administrated
with the conventional extract. From this, it has been seen that the Rhus
verniciflua stokes extract according to the present invention has an
excellent effect on inhibiting the growth of tumor.

Analysis of Antioxidative Activity of Rhus Verniciflua Stokes Extract
According to the Present Invention

[0095] In order to analyze anti-oxidative activity of the Rhus verniciflua
stokes extract according to the present invention, firstly the anticancer
activities of Rhus verniciflua stokes extract prepared by the general
method of extracting Rhus verniciflua stokes in the above Comparative
Example 1 and the Rhus verniciflua stokes extract according to the
present invention prepared from Example <1-1> were mutually
compared.

[0096] A measurement of anti-oxidative activity was performed by using a
method of measuring an electron-donating ability with DPPH method. DPPH
is an abbreviation for 1,1-diphenyl-2-picrylhydrazyl, and is widely used
a marker for measuring anti-oxidative activity due to its radical. The
testing processes were as follows: (1) 2 ml of 0.1 mM DPPH (Sigma,
D-9132) dissolved in ethanol was treated to 2 ml of sample dissolved in
ethanol; (2) it was mixed for 10 minutes, and then maintained at a dark
place for 30 minutes; (3) an absorbance was measured at 520 nm; and (4)
the degree of anti-oxidative ability (electron-donating ability, EDA) was
calculated. Vitamin E (Fluka, 95420) was used as a positive control group
and Formula was as follow.

[0098] As a result, as shown in Table 4, it has been seen that the
anti-oxidative activity of the Rhus verniciflua stokes extract with an
enhanced fisetin according to the present invention was at least 4 times
excellent as compared with the conventional Rhus verniciflua stokes
extract. It means that the anti-oxidative activity was significantly
improved by converting fustin that is plentifully included in the
conventional Rhus verniciflua stokes extract into fisetin that is an
active flavonoid.

Example 5

Preparation of Rhus Verniciflua Stokes Extract Powder with High Content of
Fisetin Using Concentration Solution

[0099] As shown in Example <1-1>, 10 kg of Rhus verniciflua stokes
was added with 10 times water to extract two times and then concentrated
to make 20 l of the concentration solution. Oxygen gas was bubbled at 0.4
VVM for 12 hours, and then the prepared Rhus verniciflua stokes extract
was concentrated to obtain 470 g of Rhus verniciflua stokes extract
powder, in which the content of fustin was 1.2% (w/w) and the content of
fisetin was 15.8% (w/w). At this time, the content ratio of
fisetin:fustin was 1:0.076.

Example 6

Preparation of Solvent Fraction with High Content of Fisetin

[0100] Each 100 g of the extracts obtained from the above Example
<1-1> and Comparative Example 1 was dissolved in 5,000 ml of water;
7,500 ml of ethyl acetate was added; then it was intensely mixed for 10
minutes using a mixer; and then it was maintained at room temperature for
separating a layer. An upper layer of ethyl acetate was collected; then
the solvent was removed; and then components were analyzed. As a result,
the content of fisetin was 42.5% (w/w) in the case of Example <1-1>
sample (referred to "A") and the content of fisetin was 7.5% (w/w) in the
case of Comparative Example 1 sample (referred to "B").

Example 7

Preparation of Fisetin Crystalline

[0101] 10 g of the solid obtained from the above Example 6 was dissolved
in 50 ml of ethanol; and then poured into cold water (cold storage) at
once. At this time, yellow crystalline of fisetin was weakly produced at
the same time. It was centrifuged at 3,000 rpm for 20 minutes to recover.
The crystalline obtained by this example was dried and then its purity
was analyzed with HPLC. As a result, the content of water was 2.8% (w/w)
and the purity of fisetin was 96.5% (w/w).

[0102] A method for preparing a Rhus verniciflua stokes extract according
to the present invention can provide Rhus verniciflua stokes extract
having high content of fisetin by converting fustin into fisetin through
a gas bubbling treatment to the conventional Rhus verniciflua stokes
extract including low content of fisetin and high content of fustin.
Accordingly, the Rhus verniciflua stokes extract having a large quantity
of fisetin according to the present invention can be commercialized as
anticancer drugs, anticancer foods, foods for preventing cancer, health
functional foods, and the like due to its excellent bioactivity. In
addition, the developments of the related industries can be induced due
to a high added-value of Rhus verniciflua stokes. Also, a method of
converting fustin into fisetin according to the present invention can be
used as a method of increasing the content of a high active flavonoid by
applying to other natural substances thereby becoming the base technology
for leading high functionalization of natural substances.

[0103] While the invention has been shown and described with reference to
certain exemplary embodiments thereof, it will be understood by those
skilled in the art that various changes in form and details may be made
therein without departing from the spirit and scope of the invention as
defined by the appended claims.

Patent applications by Sang-Jae Park, Gyeonggi-Do KR

Patent applications by Sung Pil Kwon, Seoul KR

Patent applications by Won-Cheol Choi, Incheon-Si KR

Patent applications by AZI COMPANY LTD.

Patent applications in class Bicyclo ring system having the hetero ring as one of the cyclos (e.g., chromones, etc.)

Patent applications in all subclasses Bicyclo ring system having the hetero ring as one of the cyclos (e.g., chromones, etc.)